Gene causative of Rothmund-Thomson syndrome and gene product

ABSTRACT

The RecQ 4  helicase gene, belonging to the RecQ helicase gene family, is revealed herein to be the causative gene of Rothmund-Thomson syndrome. The present inventors found out that it is possible to diagnose Rothmund-Thomson syndrome by detecting mutation of this gene. Further, they uncovered that it is possible to treat patients of Rothmund-Thomson syndrome by utilizing normal RecQ 4  helicase gene or proteins thereof.

TECHNICAL FIELD

The present invention relates to a causative gene of Rothmund-Thomsonsyndrome, methods for the diagnosis of the disease, and diagnosticagents and therapeutic agents for the disease.

BACKGROUND ART

Rothmund-Thomson syndrome ((RTS); poikiloderma congenital) is a rareautosomal recessive hereditary disease, the pathophysiology andcausative gene of which remain unrevealed. In 1868, a Germanophthalmologist, August Rothmund, reported for the first time, 10patients from an isolated village in Bayern showing crisis ofpoikiloderma at their youth and exhibiting at a high frequency juvenilecataracts (A. Rothmund, Arch. Ophthalmol. 4:159 (1887)). In 1936, anEnglish ophthalmologist, Sidney Thomson, reported 3 patients with verysimilar poikiloderma (M.S. Thomson, Br. J. Dermatol. 48:221 (1936)). Twoof the three had bone abnormality. Today, these two similar clinicalcases are recognized as Rothmund-Thomson syndrome (RTS). Many cases inchildren of a variety of races affected with this disease have beenreported worldwide, and previously over 200 cases of Rothmund-Thomsonsyndrome had been reported by Vennos et al. (E. M. Vennos et al., J. Am.Acad. Dermatol. 27:750 (1992); E. M. Vennos and W. D. James, Dermatol.Clinics. 13:143 (1995)). Although there is much clinical information onthe Rothmund-Thomson syndrome, only clinical background is available forthe diagnosis and no method for diagnosis at the laboratory level hasbeen established.

Clinical symptoms of Rothmund-Thomson syndrome include anetoderma andtelangiectasia associated with mixed hyperchromic and hypochromicregions during neonatal period, juvenile canities and alopeciaprematura, juvenile cataracts, lower stature, congenital skeletalabnormality, and increased risk of mesenchymal tumor. Cytogeneticstudies have shown that cells derived from patients withRothmund-Thomson syndrome are genetically unstable and often exhibitchromosomal recombination, and acquired somatic cell mosaicism can befound in such cells (K. L. Ying et al., J. Med. Genet. 27:258 (1990); V.M. Der Kaloustian et al., Am. J. Med. Genet. 37:336 (1990); K. H.Orstavik et al., J. Med. Genet. 31:570 (1994); M. Miozzo et al., Int. J.Cancer 77:504 (1998), N. M. Lindor et al., Clin. Genet. 49:124 (1996)).Some of the cytogenetic and clinical findings, including geneticinstability in patient cells, juvenile retardation of physical growth,skin abnormality, and high risk of tumorigenesis, are very similar tothose findings in Werner syndrome and Bloom syndrome.

Both of the causative genes of Werner syndrome and Bloom syndrome(abbreviated as WRN and BLM, respectively) belong to the RecQ DNAhelicase family, and have been identified as homologues of the E. coliRecQ gene, which encodes the DNA helicase (K. Nakayama et al., Mol. Gen.Genet. 200:266 (1985)). In addition to WRN and BLM, SGS1 from buddingyeast (S. cerevisiae) and rqh1⁺ from fission yeast (S. pombe) have beenidentified as eukaryotic homologues of E. coli RecQ DNA helicase.Mutations in the SGS1 gene are known to result in frequent homologousrecombination and non-homologous recombination in budding yeast (S.cerevisiae) cells; likewise, rqh1⁺ mutations are known to result infrequent recombination in S phase in fission yeast (S. pombe) (S.Gangloff et al., Mol. Cell. Biol. 14:8391 (1994); P. M. Watt et al.,Cell 81:253 (1995); E. Stewart et al., EMBO J. 16:2682 (1997)).

Since a trisomy mosaicism of chromosome 8 was found in two of the threeRothmund-Thomson syndrome patients examined (N. M. Lindor et al., Clin.Genet. 49:124 (1996)), the causative gene of Rothmund-Thomson syndromehas been deduced to be located on chromosome 8. However, the causativegene has not yet been identified.

DISCLOSURE OF THE INVENTION

An objective of the present invention is to identify the causative geneof Rothmund-Thomson syndrome. In addition, another objective is toprovide methods for the diagnosis of the disease as well as diagnosticand therapeutic agents for the disease. The inventors had previouslyisolated a cDNA corresponding to the RecQ4 helicase gene, belonging tothe RecQ helicase gene family (Japanese Patent Application No. Hei9-200387). The inventors considered the possibility that the RecQ4helicase gene was the causative gene of Rothmund-Thomson syndrome; theytherefore isolated the genomic DNA encoding RecQ4 helicase, andevaluated the presence of mutations in the RecQ4 helicase gene frompatients with Rothmund-Thomson syndrome by using primers prepared basedon the sequence information. The results showed that three of sevenRothmund-Thomson syndrome patients tested contained complexedheterozygotic mutations in the RecQ4 gene. Two of these patients werebrothers, and the respective mutant alleles of the two had beeninherited from the patients' family members. Aberrant transcription ofRecQ4 was specifically found in cells derived from the patients. Thissuggested that the mutations in the RecQ4 gene result in geneticinstability and are the cause of Rothmund-Thomson syndrome. In otherwords, the inventors have successfully demonstrated for the first timethat the RecQ4 gene is the causative gene of Rothmund-Thomson syndrome.

Further, from this fact, they have found that it is possible to diagnoseRothmund-Thomson syndrome by detecting mutations in the RecQ4 helicasegene; moreover, it is possible to treat the disease by compensating forthe mutations.

The present invention relates to the causative gene of Rothmund-Thomsonsyndrome, methods for the diagnosis of the disease, and diagnostic andtherapeutic agents for the disease, and more specifically to:

-   (1) a genomic DNA encoding RecQ4 helicase;-   (2) a vector comprising the genomic DNA of (1);-   (3) a host cell containing the vector of (2);-   (4) a DNA used for diagnosis of Rothmund-Thomson syndrome, which    hybridizes to a DNA encoding the RecQ4 helicase or to the expression    regulatory region thereof having a chain length of at least 15    nucleotides,-   (5) a therapeutic agent for Rothmund-Thomson syndrome, which    contains as the effective ingredient a DNA encoding RecQ4 helicase;-   (6) a therapeutic agent for Rothmund-Thomson syndrome, which    contains as the effective ingredient RecQ4 helicase;-   (7) a diagnostic agent for Rothmund-Thomson syndrome, which contains    as the effective ingredient an antibody capable of binding to RecQ4    helicase;-   (8) a method for the diagnosis of Rothmund-Thomson syndrome,    characterized by detecting mutations in the DNA encoding RecQ4    helicase or the expression regulatory region thereof;-   (9) a method for the diagnosis of Rothmund-Thomson syndrome of (8),    comprising the steps of:

(a) preparing DNA samples from patients;

(b) amplifying the prepared DNA samples by using the DNA of (4) as aprimer and determining the base sequence; and

(c) comparing the determined base sequence with that of a healthy,normal person;

-   (10) a method for the diagnosis of Rothmund-Thomson syndrome of (8),    comprising the steps of:

(a) preparing RNA samples from patients;

(b) separating the prepared RNA samples according to their size;

(c) using the DNA of (4) as a probe, hybridizing it to the separatedRNAs; and

(d) detecting hybridized RNA and comparing the results with that of ahealthy, normal person;

-   (11) a method for the diagnosis of Rothmund-Thomson syndrome of (8),    comprising the steps of:

(a) preparing DNA samples from patients;

(b) amplifying the prepared DNA samples using the DNA of (4) as aprimer;

(c) dissociating the amplified DNA into single-stranded DNAs;

(d) fractionating the dissociated single-stranded DNAs on anon-denaturing gel; and

(e) comparing the mobility of the fractionated single-stranded DNAs onthe gel with that of the healthy normal control;

-   (12) a method for the diagnosis of Rothmund-Thomson syndrome of (8),    comprising the steps of:

(a) preparing DNA samples from patients;

(b) amplifying the prepared DNA samples using oligonucleotidescomprising a base that forms a base pair with the mutated base specificfor Rothmund-Thomson syndrome in the DNA encoding RecQ4 helicase, or theexpression regulatory region thereof, as at least one of the primers;and

(c) detecting the amplified DNA fragment;

-   (13) a method for the diagnosis of Rothmund-Thomson syndrome of (8),    comprising the steps of: (a) preparing DNA samples from patients;

(b) amplifying the prepared DNA samples using a pair of DNA of (4),which is prepared so as to flank the mutated base specific toRothmund-Thomson syndrome, as the primer;

(c) hybridizing to the amplified product a pair of oligonucleotidesselected from the group of:

(i) an oligonucleotide synthesized such that the base forming a basepair with the mutated base in the amplified product corresponds to the3′-terminus, and an oligonucleotide synthesized such that theneighboring (on the 3′ side) base to said 3′-terminus corresponds to the5′ terminus;

(ii) an oligonucleotide synthesized such that the base forming a basepair with the base of a normal healthy person which corresponds to themutated base in the amplified product corresponds to the 3′-terminus,and an oligonucleotide synthesized such that a neighboring (on the 3′side) base to said 3′-terminus corresponds to the 5′-terminus;

(iii) an oligonucleotide synthesized such that the base forming a basepair with the mutated base in the amplification product corresponds tothe 5′-terminus, and an oligonucleotide synthesized such that theneighboring (on the 5′ side) base to said 5′-terminus corresponds to the3′-terminus;

(iv) an oligonucleotide synthesized such that the base forming a basepair with the base of a normal healthy person which corresponds to themutated base in the amplified product corresponds to the 5′-terminus,and an oligonucleotide synthesized such that the neighboring (on the 5′side) base to said 5′-terminus corresponds to the 3′ terminus

(d) ligating the oligonucleotides; and

(e) detecting the ligated oligonucleotides; and

-   (14) a method for the diagnosis of Rothmund-Thomson syndrome of (8),    comprising the steps of:

(a) preparing protein sample from patients;

(b) contacting an antibody against RecQ4 helicase with the preparedprotein sample;

(c) detecting proteins binding to the antibody.

The present invention primarily relates to the causative gene ofRothmund-Thomson syndrome (RTS). The inventors have found that thecausative gene of Rothmund-Thomson syndrome encodes human RecQ4helicase. The base sequences of the genomic DNA encoding RecQ4 helicasedetermined by the inventors are shown in SEQ ID NO: 1 (expressionregulatory region) and SEQ ID NO: 2 (exon and intron regions).

The genomic DNA encoding RecQ4 helicase of the present invention can beobtained by using the entire base sequence described in any of SEQ IDNOs: 1-3, or a part thereof, as a hybridization probe to screen agenomic DNA library. Alternatively the DNA can be amplified and isolatedby polymerase chain reaction (PCR) using a genomic DNA or genomic DNAlibrary as the template and using as the primer a part of the basesequence described in SEQ ID NO: 1 or 2.

The genomic DNA of the present invention, as described below, can beused to prepare primers and probes for the diagnosis of Rothmund-Thomsonsyndrome, to treat Rothmund-Thomson syndrome by gene therapy, and toproduce RecQ4 helicase.

The present invention also relates to DNA hybridizing to DNA encodingRecQ4 helicase, or the expression regulatory region thereof, whichcomprises at least 15 nucleotides and is used for the diagnosis ofRothmund-Thomson syndrome. Preferably, this DNA hybridizes specificallyWith a DNA encoding RecQ4 helicase or the expression regulatory regionthereof.

The term “hybridizing specifically with” herein means that there is nosignificant cross-hybridization with DNAs or RNAs encoding otherproteins under usual hybridization conditions, preferably understringent hybridization conditions. Such a DNA doesn't have to becompletely complementary to the target sequence but is generally atleast 70%, preferably at least 80%, and more preferably at least 90%(for example, 95% or more) identical to the target at the base sequencelevel.

When used as a primer, the oligonucleotide is generally a 15mer-35mer,preferably a 20mer-28mer.

The primer may be any one of the above, so long as it is capable ofamplifying at least a part of the coding region of RecQ4 helicase or aregion regulating expression thereof. Such a region includes, forexample, the exon region, the intron region, the promoter region and theenhancer region of the RecQ4 helicase gene.

On the other hand, if the oligonucleotide probe is synthetic, itgenerally consists of at least 15 bases or more. It is possible to use adouble-stranded DNA obtained from a clone inserted into a vector, suchas plasmid DNA, as a probe, as well as RNA synthesized from the clone bytranscription. The region used as a probe can be any region so long asit hybridizes specifically to at least a part of the coding region ofRecQ4 helicase or the region regulating expression thereof. Such aregion to which the probe hybridizes includes, for example, the exonregion, the intron region, the promoter region and the enhancer regionof the RecQ4 helicase gene.

Probes such as oligonucleotides, double-stranded DNAs, and RNAs can beused with proper labels. Labeling methods include, for example, endlabeling for oligonucleotides, random primer labeling or PCR method fordouble-stranded DNAs, and in-vitro transcription labeling for RNAs.Compounds useful for labeling include [γ-³²P] ATP for end labeling,[α-³²P] dCTP or digoxigenin (DIG)-dUTP for random primer labeling andPCR method, and [α-³²P] CTP or DIG-UTP for in-vitro transcriptionlabeling.

The “diagnosis of Rothmund-Thomson syndrome”, in accordance with thepresent invention, is characterized by the detection of mutations in theRecQ4 helicase gene. The “diagnosis of Rothmund-Thomson syndrome” inaccordance with the present invention includes not only testing ofpatients exhibiting symptoms of Rothmund-Thomson syndrome due tomutations in the RecQ4 helicase gene, but also includes testing to judgewhether or not the subjects are potentially affected withRothmund-Thomson syndrome due to the mutations in the RecQ4 helicasegene.

In addition, “detection of mutations in the RecQ4 helicase gene”, inaccordance with the present invention, includes both detection at theprotein level and detection at the DNA and at the RNA levels.

One embodiment of the diagnostic test method, in accordance with thepresent invention, is a method for directly determining base sequence ofthe RecQ4 helicase gene from patients. This method comprises the stepsof: (a) preparing DNA samples from patients; (b) amplifying prepared DNAsamples derived from patients by using the DNA of the present inventionas a primer to determine the base sequence; and (c) comparing thedetermined base sequence with that of a healthy normal person. Directdetermination of base sequence includes direct determination of basesequence of RecQ4 genomic DNA and direct determination of base sequenceof RecQ4 cDNA.

When the base sequence of genomic DNA of RecQ4 is intended to bedetermined directly, the genomic DNA is prepared from patients, and theRecQ4 gene is amplified from the genomic DNA from patients by using asense primer and an antisense primer specific to the RecQ4 gene. It ispreferred that the primers are 20mer-28mer in length and that the Tmvalues thereof are within the range of 65° C.-75° C. in theamplification of the RecQ4 gene. The RecQ4 genomic DNA, amplified usinga sense primer and an antisense primer, is preferably 1 kb-1.5 kb inlength. It is preferable to design the sense primer and antisense primerso that the 50 bp-100 bp 5′ and 3′ ends of the RecQ4 genomic DNAfragment to be amplified overlap with other genomic DNA fragments,thereby covering the entire region of about 6.5 kb of RecQ4 genomic DNA.Further, the expression regulatory region of the RecQ4 gene may beamplified and used as a test subject. The base sequence determination ofthe amplified fragment can be performed, for example, by the PCR-basedmethod of Hattori et al. (Electrophoresis 13, pp 560-565 (1992)).Specifically, the reaction is carried out using a PRISM sequencing kitcontaining fluorescent dideoxy-terminator (Perkin-Elmer), and usingspecific primers to the fragment of RecQ4 genomic DNA to be amplified.Subsequently, the base sequence is determined by an automatic sequencerfrom Applied Biosystems (Model ABI 373), and the data is analyzed by anattached Macintosh computer. The judgment on the presence of mutationscan be formed, for example, by analyzing the base sequence, as a seriesof peaks of waveforms with four colors by using analytical software forbase sequence such as Sequencing Analysis (Applied Biosystems). That is,mutations can be detected by comparing the series of peaks of waveformsrepresenting base sequence of genomic DNA of the normal RecQ4 gene withthe series of peaks of waveforms representing base sequence of genomicDNA of a patient's RecQ4 gene. Further, the judgment can be formedthrough sequence analysis, using base sequence-editing software such asDNASIS. In other words, mutations can be detected by comparing thesequence of normal RecQ4 genomic DNA with the sequence of genomic DNARecQ4 from patients with a computer.

In the case where the base sequence of RecQ4 cDNA is determineddirectly, the cDNA is prepared from the RNA sample of patients byreverse transcription, and then the RecQ4 gene is amplified frompatients using the sense primer and the antisense primer specific to theRecQ4 gene. It is preferable that the primers are 20 mer-28 mer inlength and the Tm values thereof are within the range of 65° C.-75° C.in the amplification of the RecQ4 gene. The RecQ4 cDNA amplified usingthe sense primer and antisense primer is preferably 1 kb-1.5 kb inlength. It is preferable to design the sense primer and antisense primersuch that the 50 bp-100 bp of 5′ and 3+ ends of the RecQ4 genomic DNAfragment to be amplified overlap with other genomic DNA fragments, andcover the entire region of RecQ4 cDNA which is about 4 kb. The basesequence determination of the amplified fragment can be performed in thesame manner as described above for genomic DNA, for example, by thePCR-based method of Hattori et al. (Electrophoresis 13, pp 560-565(1992)). Specifically, the reaction is carried out using a PRISMsequencing kit containing fluorescent dideoxy-terminator (Perkin-Elmer);in which specific primers are used to the fragment of RecQ4 cDNA to beamplified. Subsequently, the base sequence is determined by an automaticsequencer from Applied Biosystems (Model ABI 373), and the data isanalyzed by an attached Macintosh computer. The judgment on the presenceof mutations can be formed, for example, by analyzing the base sequenceas a series of peaks of waveforms with four colors by using analyticalsoftware for base sequence such as Sequencing Analysis (AppliedBiosystems). That is, mutations can be detected by comparing the seriesof peaks of waveforms representing the base sequence of genomic DNA ofthe normal RecQ4 gene with the series of peaks of waveforms representingthe base sequence of genomic DNA of the patients' RecQ4 gene. Further,the judgment can be formed through sequence analysis, usingsequence-editing software such as DNASIS. In other words, mutations canbe detected by comparing the cDNA sequence of normal RecQ4 with the cDNAsequence of RecQ4 from patients with a computer.

The method for the diagnosis of the present invention includes a varietyof other methods in addition to the direct determination method for thebase sequence derived from patients as described above. In oneembodiment, such a method comprises the steps of: (a) preparing DNAsamples from patients; (b) amplifying the prepared DNA samples derivedfrom patients using the DNA of the present invention as a primer; (c)dissociating the amplified DNA into single-stranded DNAs; (d)fractionating the dissociated single-stranded DNAs on a non-denaturinggel; and (e) comparing the mobility of the dissociated single-strandedDNAs on the gel with that of the DNAs from a healthy normal person.

Such a method includes the method of PCR-SSCP (single-strandconformation polymorphism). The PCR-SSCP method is designed based on theprinciple that two single-stranded DNAs, of which lengths are identicalbut which base sequences are different, form distinct higher-orderstructures through their respective intramolecular interactions andtherefore show different electrophoretic motilities to each other. Thatis, the higher-order structure of a single-stranded DNA with amutation(s) is different from that of a single-stranded DNA withoutmutation(s), and thus the two exhibit different electrophoreticmotilities on a non-denaturing gel. This difference makes it possible todetect the mutation(s) (Orita et al., Proc. Natl. Acad. Sci. USA, 1989,vol. 86, pp 2766-pp 2770).

The PCR-SSCP method can be used to detect alterations in the sequence ofRecQ4 genomic DNA or RecQ4 cDNA. When mutations are intended to bedetected in RecQ4 genomic DNA, the RecQ4 gene is amplified from each ofthe genomic DNAs of healthy normal person and patient, using a senseprimer and an antisense primer specific to the RecQ4 gene. In thisexperiment, the primers are previously radiolabeled with ³²P by an endlabeling method. It is preferred that the length of primer is 20 mer-28mer and the Tm value is within the rage of 65° C.-75° C. Further, it ispreferable that the RecQ4 genomic DNA to be amplified, using the senseprimer and antisense primer, is 300 bp or shorter in length. Preferably,the sense primer and the antisense primer are designed so that the 60bp-100 bp of 5′ and 3′ ends of the RecQ4 genomic DNA fragment to beamplified overlap with other genomic DNA fragments, and cover the entireregion of RecQ4 genomic DNA, which is about 6.5 kb. The amplified DNAfragment is electrophoresed on a 5% non-denaturing polyacrylamide thethickness and the length of which is 0.3 mm-0.35 mm and 40 cm,respectively. The gel is analyzed by autoradiography and the mobility ofthe band from the patient is compared with that from a healthy normalperson for detection of mutations.

When mutations are intended to be detected in RecQ4 cDNA, the cDNA isprepared from a patient's RNA sample by reverse transcription, and theRecQ4 gene is amplified from cDNAs of healthy normal person and patientusing a sense primer and an antisense primer specific to the RecQ4 gene.In this experiment, the primers are previously radiolabeled with ³²P byan end labeling method. It is preferred that the length of primer is20mer-28mer and the Tm value is within the rage of 65° C.-75° C.Further, it is preferred that the RecQ4 cDNA to be amplified using thesense primer and antisense primer is 300 bp or shorter in length.Preferably the sense primer and antisense primer are designed so thatthe 60 bp-100 bp of 5′ and 3′ ends of the RecQ4 cDNA fragment to beamplified overlap with other cDNA fragments and cover the entire regionof RecQ4 cDNA which is about 4 kb. The amplified DNA fragment iselectrophoresed on a 5% non-denaturing polyacrylamide the thickness andthe length of which is 0.3 mm-0.35 mm and 40 cm, respectively. The gelis analyzes by autoradiography and the mobility of the band from thepatient is compared with that from a healthy normal person for detectionof mutations.

The above-described methods for diagnosis are just a few specificexamples and those skilled in the art may properly modify the detailedprocedures of the methods. In a test of the genomic DNA, the presence ofmutations can be tested in the expression regulatory region (promoterregion and enhancer region). Moreover, to test if a particular region ofgenomic DNA or cDNA has a mutation, a DNA fragment containing the siteto be tested may be prepared and used for the test instead of a DNAcovering the entire region of the RecQ4 gene.

Alternatively, RNA, instead of DNA prepared from patients, can also beused for the detection. Such a method comprises the steps of: (a)preparing RNA samples from patients; (b) separating the prepared RNAsamples based on their size; (c) allowing the DNA probe of the presentinvention, which has been detectably labeled, to hybridize with theseparated RNA; and (d) detecting the hybridized RNA and comparing theRNA with that from a healthy normal person. In a specific example, theRNA prepared from patients is electrophoresed, and Northern blotting isperformed using the probe DNA of the present invention to detect thepresence and intensity of the signal, and/or the difference in mobilityon a gel.

In addition to these methods, it is possible to perform the test of thepresent invention by detecting mutations at positions selectedpreviously.

One embodiment of such a test method comprises the steps of: (a)preparing DNA samples from patients; (b) amplifying the prepared DNAsamples from patients using an oligonucleotide containing a base capableof forming a base pair with the mutated base specific forRothmund-Thomson syndrome in the DNA encoding RecQ4 helicase or theexpression regulatory region thereof as at least one of the primers; and(c) detecting the amplified DNA fragment.

Such a method includes, for example, the method of MASA(mutant-allele-specific amplification) (Matsumoto et al., ExperimentalMedicine 15:2211-2217 (1977); Unexamined Published Japanese PatentApplication (JP-A) No. Hei 10-201498).

MASA is a method in which template genomic DNA or cDNA is amplified bypolymerase chain reaction (PCR) using oligonucleotides containing basescapable of forming a base pair with the mutated base as one of theprimers, and subsequently subjecting them to gel electrophoresis todetect the mutant alleles.

To conduct this method in accordance with the present invention, a pairof primers (5′-side sense primer and 3′-side antisense primer) issynthesized to amplify the template DNA. Herein, the 5′-side senseprimer is synthesized so as to contain a base capable of forming a basepair with the mutated base. The 5′-side sense primer is designed so asto function as a specific primer when a mutation-containing DNA encodingRecQ4 helicase or the expression regulatory region thereof is used as atemplate, but not when a mutation-free DNA encoding RecQ4 helicase orthe expression regulatory region thereof is used. In this case, it ispreferred that the base forming a base pair with the mutated base isplaced at the 3′ end of the 5′-side sense primer. On the other hand, anoligonucleotide primer specifically hybridizing to the region without amutation is used as the 3′-side sense primer. Polymerase chain reactionis carried out under a condition where the amplification is veryefficient, due to the efficient hybridization of the 5′-side senseprimer to the template of mutation-containing DNA fragment (abnormalallele), and where the efficiency of amplification is extremely low, dueto the incompetence of the 5′-side sense primer in the hybridization tothe template of mutation-free DNA fragment (normal allele).

For example, heating once at 95° C. for 5 minutes; heating at 94° C. for30 seconds, heating at 50° C. for 30 seconds and heating at 72° C. for30 seconds as one cycle, and that for 40 cycles; and a heating at 72° C.for 4 minutes are carried out.

Alternatively, polymerase chain reaction can be performed in the samemanner, using a 3′-side antisense primer containing a base forming abase pair with the mutated base and a 5′-side sense primer that is anoligonucleotide specifically hybridizing to the region without amutation.

Thus mutation-containing sample DNA can be amplified efficiently becausethe DNA can hybridize to the mutation-containing primer. For example,when the amplified DNA is subjected to electrophoresis, it can bedetected as a positive band on the gel. On the other hand, sample DNAfrom a normal subject is incompetent in the hybridization with a primercontaining the mutation and as a result the amplification is notachieved and no band is observed on the gel.

Further, in addition to the detection with the above-mentionedmutation-containing primer, another detection can be carried out using aprimer without the mutation (which contains a base incapable of forminga base pair with the mutated base but capable of forming a base pairwith the normal base) corresponding to the primer above, to judgewhether the subject has the mutation homozygously or heterozygously.That is, when a band is detected with the mutation-containing primer andno band is detected with the mutation-free primer, then the sample DNAcan be judged to have the homozygous mutation associated withRothmund-Thomson syndrome. Alternatively, when a band is observed withboth of the two primers, then the sample DNA can be judged to have themutation heterozygously, or when a band is detected merely with theprimer without the mutation, then the DNA can be judged as normal inrespect to the tested site.

Another embodiment of the method for diagnosis of the present inventioncomprises the steps of: (a) preparing DNA samples from patients; (b)amplifying the prepared DNA samples from patients using oligonucleotidesprepared as a pair to flank a mutated base specific to Rothmund-Thomsonsyndrome as a primer; (c) hybridizing to the obtained amplificationproduct any pair of the oligonucleotides of: (i) an oligonucleotidesynthesized such that the 3′-terminus thereof corresponds to the baseforming a base pair with the mutated base in the amplification product,and an oligonucleotide synthesized such that the neighboring base. (onthe 3′ side) to said 3′-terminus is placed at the 5′-terminus of thesynthesized oligonucleotide; (ii) an oligonucleotide synthesized suchthat the 3′-terminus thereof corresponds to the base forming a base pairwith the base from a healthy normal person corresponding to a mutatednucleotide in the amplification product, and an oligonucleotidesynthesized such that the neighboring base (on the 3′ side) to said3′-terminus is placed at the 5′-terminus of the synthesizedoligonucleotide; (iii) an oligonucleotide synthesized such that the5′-terminus thereof corresponds to a base forming a base pair with themutated base in the amplification product, and an oligonucleotidesynthesized such that the neighboring base (on the 5′ side) to the5′-terminus is placed at the 3′-terminus of the synthesizedoligonucleotide; (iv) an oligonucleotide synthesized such that the5′-terminus thereof corresponds to a base forming a base pair with thebase from patients corresponding to the mutated base in theamplification product, and an oligonucleotide synthesized such that theneighboring base (on the 5′ side) to the 3′-terminus is placed at the5′-terminus of the synthesized oligonucleotide; ligating theseoligonucleotides; and (d) detecting the ligated oligonucleotides.

Such a detection method includes, for example, OLA (OligonucleotideLigation Assay) (Matsumoto et al., Experimental Medicine 15:2211-2217(1977); JP-A No. Hei 10-201498). First, primers are designed to beplaced upstream and downstream of each site to be detected (i.e., sitespredicted to contain a mutation) with an appropriate spacing, and thenpolymerase chain reaction is conducted to amplify genomic DNA fragmentor cDNA fragment containing the site to be detected. The distancebetween each site to be detected and the primer can be selectedarbitrarily, but 100 bp-200 bp is preferred. Further, there is noparticular limitation on the number of nucleotides in the primer, but aprimer of 20mer-30mer is preferred.

On the other hand, based on the base sequence of the RecQ4 helicasegene, an oligonucleotide consisting of 18-30 nucleotides is synthesizedso that the above-mentioned site to be detected is placed at the 3′ endthereof and that a base forming a base pair with the predicted mutatedbase is placed at the 3′ end (the synthesized oligonucleotide isreferred to as “oligonucleotide A”). Further, another oligonucleotideconsisting of 18-30 nucleotides is synthesized so that the baseneighboring (on the 3′ side) to the above-mentioned site to be detectedcorresponds to the 5′ end thereof (the synthesized oligonucleotide isreferred to as “oligonucleotide X”). The mutant-type primer can beprepared by mutagenizing the normal sequence using known technique(e.g., by using a mutagenesis kit (In vitro Mutagenesis Kit, TaKaRaShuzo)), or alternatively chemically synthesizing the primer based onthe sequence designed with a mutation.

According to this preparation, for the convenience of purification anddetection of the oligonucleotides ligated through the ligase reaction asdescribed below, it is preferable, for example, to label the 5′ end ofoligonucleotide A with biotin or the like, to label the 3′ end ofoligonucleotide X with digoxigenin-11-dideoxy UTP or the like, and toadd a phosphate group to the 5′ end.

Then, oligonucleotides A and X are annealed with the above-mentionedproduct of polymerase chain reaction to ligate oligonucleotides A and Xwith each other. When a mutation of interest is present in the sampleDNA, the 3′-end of oligonucleotide A can form a base pair with themutated base and as a result oligonucleotide A can be connected tooligonucleotide X; and this allows the production of oligonucleotideswith labels at both ends (for example, biotin and digoxigenin).

For example, if the product has biotin and digoxigenin at either ends,then the mutation is detected from an arising color reaction, stemmingfrom the absorbance of the product on a plate coated with streptavidinand the subsequent reaction with an anti-digoxigenin antibody conjugatedwith alkaline phosphatase or the like.

On the contrary, when the sample DNA does not contain the mutation, thenthe 3′-end of oligonucleotide A cannot form a base pair with thecorresponding base in the template DNA, and as a consequence,oligonucleotides A and X cannot be connected with each other.

Accordingly, even when oligonucleotides A and X are labeled, forexample, with biotin and digoxigenin, respectively, oligonucleotideswith respective labels at respective ends are not formed; and thus evenwhen the ligation reaction product is bound to the plate coated withavidin and the anti-digoxigenin antibody conjugated with alkalinephosphatase or the like is allowed to react thereto, no color reactionis detectable (Delahunty et al., Am. J. Hum. Genet. 58: 1239-1246,1996).

Further, when an oligonucleotide as described below, specificallydetecting DNA that doesn't contain mutations at the site to be testedfor detection is used, it is possible to judge whether or not thesubject has the mutation homozygously. Specifically, an oligonucleotidecontaining the normal sequence, that has no mutated nucleotide at theabove-mentioned site to be detected (which is referred to asoligonucleotide B), is synthesized in the same manner as oligonucleotideA and then the ligation assay between oligonucleotide B andoligonucleotide X is performed in addition to the ligation assay witholigonucleotide A and oligonucleotide X.

If the experimental result shows a positive color reaction witholigonucleotides A and X but not with oligonucleotides B and X, thesample DNA can be judged to have a homozygous mutation associated withRothmund-Thomson syndrome. Alternatively, when color development isdetected in either assays with oligonucleotides A and X and witholigonucleotide B and X, the DNA can be judged to have a heterozygousmutation; when the color reaction is positive in the assay witholigonucleotides B and X alone, the DNA is judged normal at the testedsite.

Alternatively, the mutation can be detected in the same manner as withthe above-mentioned oligonucleotides A and X, by the combined use of anoligonucleotide in which a base forming a base pair with the predictedmutated base has been introduced at the 5′ end and an oligonucleotideprepared such, so that the base flanking (on the 5′ side) to theabove-mentioned site to be detected corresponds to the 3′ end thereof.

The detection method of the present invention can also be conducted byusing antibody capable of binding to RecQ4 helicase. In one embodiment,such a method comprises the steps of: (a) preparing protein sample frompatients; (b) contacting antibody against RecQ4 helicase with theprepared protein; and (c) detecting a protein binding to the antibody.

The antibody to be used in the test of the present invention may be amonoclonal antibody or a polyclonal antibody. Antibodies binding toRecQ4 helicase can be prepared by a method known to those skilled in theart (see, for example, Japanese Patent Application No. Hei 9-200387).The antigens utilized to prepare antibodies can be provided, forexample, by introducing the gene encoding the antigen into anappropriate plasmid vector and expressing the gene product in E. colior, alternatively, by introducing the gene into a baculovirus vector andexpressing the gene product in insect cells. Alternatively, a syntheticpeptide can also be used. The expression vector can be, for example, avector such as pQE30 (Qiagen.) in the case of E. coli expression, or abaculovirus vector such as pAcHLT-B (PharMingen). In this case, thepurification of the gene product can be simplified by attaching a tag,such as Flag (Chiang, C. et al., EMBO J., 12: 2749-2762 (1993)) or 6xhis(Immunol. Meth. 4: 121-152 (1990)), to the product. The expressed geneproduct can be purified utilizing the tag.

A number of cases have been discovered where a protein, which has atruncation at the C terminus of the normal RecQ4 helicase, is presumedto be produced by frame shift or a newly generated termination codon dueto mutations in the RecQ4 helicase gene in patients withRothmund-Thomson syndrome (see Examples). Accordingly, it is possible tocarry out easily and efficiently the diagnosis of Rothmund-Thomsonsyndrome by using antibody recognizing the C terminus of RecQ4 helicase(see Japanese Patent Application No. Hei 10-311284).

In addition, when another antibody recognizing the N terminal region ofRecQ4 helicase is used in conjunction with the antibody recognizing theC terminal region in the test of the present invention, it is possibleto test which of the two, namely an aberrant expression or structuralabnormality of the causative gene, is the cause of the diseaseassociated with mutations of the RecQ4 helicase gene in patients. Thatis, it is believed that when mutations are generated in the causativegene of the disease, caused by the mutation of the RecQ4 helicase,translation products without the normal C terminus are apt to beproduced, due to the resulting frame shift and generation of atermination codon. Therefore, mutations are considered to occurfrequently in the C terminal region while the N terminal region isnormal. Thus, there is a high possibility that the translation productfrom the causative gene is detectable by antibody against the N terminalregion but not by antibody against the C terminus region when there is astructure abnormality in the translation product.

Furthermore, for example, it has been known that, in the WRN helicasegene, the expression level of mRNA corresponding to the gene containinga mutation(s) is markedly reduced (Yamabe, Y. et al., Biochem. Biophys.Res. Commun., 236: 151-154 (1997)). In the RecQ4 helicase gene, thelevel of mRNA corresponding to the gene was indeed significantly reducedin RTS patients (see Examples). In such cases, it can be expected thatthe translation product per se from the RecQ4 helicase gene containingmutations is sometimes undetectable. In such aberrant expression of theRecQ4 helicase gene (marked reduction of the expression), it is expectedthat no immunological reaction is detectable by any antibody.Accordingly, the diagnosis of Rothmund-Thomson syndrome can be conductedby the combined use of these two antibodies.

The test, using antibody binding to RecQ4 helicase of the presentinvention, can be conducted utilizing a variety of publicly knownimmunological techniques. A preferred method is Western blotting.Specifically, cells from a patient are lysed in a buffer containingdetergent, the resulting sample is electrophoresed on an SDSpolyacrylamide gel (SDS-PAGE) containing sodium dodecyl sulfate (SDS),the proteins are transferred onto a filter from the gel, the protein ofinterest can be detected on the filter by using antibody binding toRecQ4 helicase. It is also possible to detect RecQ4 helicase by ELISA(enzyme-linked immunosorbent assay, ELISA; I. Roitt et al., In“Immunology”, The C. V. Mosby Co., 1989, pp 25.5-25.6) or byimmunohistochemical staining on tissue sections. The antibody can belabeled, for example, with an enzyme label such as alkaline phosphataseor horseradish peroxidase. In this case, the protein of interest can bedetected through color reaction. In addition, a fluorescent label can bealso utilized. The label can also be linked to a secondary antibodyrecognizing the antibody against the protein of interest for thedetection of the protein of interest. Alternatively, the label can alsobe linked to the antibody against the protein of interest for thedetection. By utilizing the above-mentioned method, it is possible toconduct the test for the lack, accumulation or abnormal cellulardistribution of RecQ4 helicase.

Thus, the antibody binding to RecQ4 helicase can be used in thediagnosis of Rothmund-Thomson syndrome. When used as a diagnostic agent,the antibody is generally used in a buffer of about pH6-pH8 (forexample, phosphate buffer, HEPES buffer, or Tris buffer), and ifrequired, it can be mixed with a carrier (for example, bovine serumalbumin of about 1-5% or gelatin of about 0.2%), a preservative (forexample, 0.1% sodium azide), and so on.

Samples from patients used in the diagnosis of the present invention canbe, if it is a test of genomic DNA, any cells containing genomic DNAderived from patients, and, if it is a test of RNA, cDNA or protein, inprinciple any cells can be used as far as the cells are derived from thepatient and correspond to cells expressing the RecQ4 helicase gene in ahealthy normal person. For example, it is possible to use fibroblastcells established from a piece of skin tissue obtained by biopsy, cellsprepared by transforming B. lymphocytes contained in leukocytes obtainedby blood collection by using Epstein-Barr virus, or the like.

The present invention further relates to a therapeutic agent forRothmund-Thomson syndrome. In one embodiment, such a therapeutic agentcomprises a DNA encoding RecQ4 helicase as the effective ingredient. Ifa DNA encoding RecQ4 helicase is used as the therapeutic agent,full-length genomic DNA encoding RecQ4 helicase or a part thereof, orfull-length RecQ4 helicase cDNA (cDNA encoding human RecQ4 helicase isshown in SEQ ID NO: 3) or a part thereof is introduced into anappropriate vector, such as adenoviral vector, retroviral vector, or thelike, and then, the resulting DNA is administered intravenously orlocally to the diseased site to the patient. The administration methodcan include an ex-vivo method as well as in-vivo method.

Thus, the RecQ4 helicase gene containing the mutations can be replacedwith the normal gene in the patient, or alternatively the normal genecan be administered to the patient in an additional fashion, therebytreating Rothmund-Thomson syndrome.

In another embodiment associated with the therapeutic agent forRothmund-Thomson syndrome, RecQ4 helicase is used as an activeingredient. RecQ4 helicase can be prepared as a naturally occurringprotein, or as a recombinant protein provided by genetic recombinationtechniques. The amino acid sequence of human RecQ4 helicase is shown inSEQ ID NO: 4. The naturally occurring protein can be isolated fromtissues or cells highly expressing RecQ4 helicase (for example, thymusand testis, chronic myelogenous leukemia K562 cell, promyelocyticleukemia HL-60cells, HeLa cell) by a method well known to those skilledin the art, for example, affinity chromatography using antibody againstRecQ4 helicase. On the other hand, it is possible to prepare therecombinant protein, for example, through culturing cells transformedwith DNA encoding RecQ4 helicase (for example, SEQ ID NO: 3). Cells usedfor the production of the recombinant protein include mammalian cells,insect cells, yeast cells, and E. coli. The expression vectors to beused are known to those skilled in the art. Introduction of the vectorinto host cells and purification of the recombination protein from theresulting transformants can be achieved by using methods known to thoseskilled in the art. When it is intended to use the obtained RecQ4helicase as the therapeutic agent for Rothmund-Thomson syndrome, theRecQ4 helicase can be administered directly or alternativelyadministered after formulating the RecQ4 helicase by a publicly knownpharmaceutical production method. For example, the protein can beadministered by dissolving the protein into a commonly usedpharmaceutical medium, e.g., a neutral solution such as PBS. The dosagedepends on various factors, such as the patient's body weight, age,health, and the type of administration method to be used. Those skilledin the art can properly select a suitable dosage. The administration canbe performed, for example, subcutaneously, orally, directly to thedisease site, etc.

In another embodiment associated with the therapeutic agent forRothmund-Thomson syndrome, the agent comprises compound capable ofstimulating and elevating the expression of RecQ4 helicase as theeffective ingredient.

There is the possibility that the onset of Rothmund-Thomson syndrome isclosely associated with the reduction of the expression level of theRecQ4 helicase gene. Accordingly, stimulating and elevating theexpression of the RecQ4 helicase gene may treat Rothmund-Thomsonsyndrome.

A compound capable of stimulating and elevating the expression of theRecQ4 helicase gene can be obtained by inserting the regulatory region(promoter region and enhancer region) responsible for the expression ofthe RecQ4 helicase gene into a vector containing luciferase as areporter, introducing the resulting DNA construct into cultured cells,and screening the cells with the introduced DNA for the compoundstimulating and elevating the luciferase activity. The base sequence ofthe expression regulatory region of the human RecQ4 helicase gene isshown in SEQ ID NO: 1. A reporter gene that can be used for this purposeincludes the luciferase gene from firefly and the luciferase gene fromRenilla. Vectors containing these reporter genes include fireflyluciferase reporter vector pGL3 and Renilla luciferase reporter vectorpRL (Promega). The cells to which the DNA is introduced include human293 cell, HeLa cell, K562 cell and monkey COS7 cell. Using a publiclyknown method, such as calcium phosphate precipitation method, liposomemethod, and electroporation method, introduction of the DNA into cellscan be performed. When the method is conducted in accordance with thepresent invention, the reporter gene connected with the promoter regionof the human RecQ4 helicase gene is introduced into human or monkeyculture cells by the methods as described above and then the cells arecultured. Each of the various types of sample to be tested are added tothe culture medium during the culture and then cell extract is prepared48 hours after the addition of the compound; the luciferase activity ina cell extract is detected by a method as describe in a reference(Yamabe et al., Mol. Cell. Biol., 1998, vol. 18, pp 6191-pp 6200).Compounds capable of stimulating and elevating the expression of theRecQ4 helicase gene can be identified through the procedures describedabove. The sample to be tested in the screening includes, for example,cell extract, expression products from gene library,low-molecular-weight synthetic compound, synthetic peptide, naturalcompound, etc., but is not limited to these examples.

As with the case of the above-mentioned RecQ4 helicase used as atherapeutic agent, when a compound stimulating and elevating theexpression of the RecQ4 helicase gene is used as a therapeutic agent forthe disease, it can be administered after formulating the compound by apublicly known pharmaceutical production method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(a) shows a family tree of patients with Rothmund-Thomson syndromeand other members of the family. “I” represents parents; “1” indicatesfather and “2” indicates mother. Each of half-closed square and circleindicates a genetic carrier with a mutation in one allele of the RecQ4.“II” represents brothers or sisters (1-6) of the patients. Each ofcompletely closed square (II.3, male) and circle (II.6, female)represents a patient with Rothmund-Thomson syndrome who has mutations inboth alleles of the RecQ4 gene. II.2, II.4, and II.5 were not patientsaffected with Rothmund-Thomson syndrome and therefore no analysis wasperformed for them. The person II.1 indicated by the shaded symbol hadbeen diagnosed as a patient with Rothmund-Thomson syndrome based on theclinical findings.

FIG. 1(b) shows the results of analysis for the mutation in the RecQ4gene in the patients with Rothmund-Thomson syndrome and their parents.Lane I.1 represents the father; lane I.2, the mother; lane II.3, patientII.3; lane II.6, patient II.6. Based on the results, it has beenrevealed that the mother has a 7-base deletion (mut-1) in one allele ofthe gene inherited from her parent.

FIG. 2 shows the results of direct base sequencing analysis for theRecQ4 gene in its mutational region.

(a) shows base sequences of the region comprising mut-1 (residue1641-1672 in the protein-coding region) in normal and mutant RecQ4genes. The region encircled by mut-1 (7-base deletion) was amplified byPCR using genomic DNAs prepared from a healthy normal person andpatients II.2 and II.6 with Rothmund-Thomson syndrome, to analyze thebase sequences. The results of sequencing of normal and mutant sequencesare indicated below.

(b) shows base sequences of the region comprising mut-2 (residue2257-2280 of the protein-coding region) in normal and mutant RecQ4genes. The region encircled by mut-2 (point mutation from C to T) wasamplified by PCR using genomic DNAs prepared from a healthy normalperson and patients II.2 and II.6 with Rothmund-Thomson syndrome. Thesequencing analysis was carried out in the same manner as in (a).

FIG. 3 shows a schematic illustration of deleted RecQ4 helicasemolecules generated by mut-1 to mut-4. The term “normal” represents thefull-length RecQ4 helicase, consisting of the 1208 amino acids deducedfrom the coding region of the cloned RecQ4 gene. The shaded regionrepresents a helicase domain that is conserved in all RecQ helicases.

FIG. 4 shows the investigated results of down-regulated expression ofthe RecQ4 gene in cells from patients with Rothmund-Thomson syndrome.The transcripts of the RecQ4 gene from cells derived from patients withRothmund-Thomson syndrome were compared with those from a healthy normalperson. Poly(A)⁺ RNAs from skin fibroblast cells were prepared frompatients with Rothmund-Thomson syndrome with mutations in the RecQ4 gene(II.3 and AG05013), from three other patients with Rothmund-Thomsonsyndrome (AGO5139and AG03587A from NIA, Aging Cell Repository; andTC4398 provided by Dr. R. Miller) who had no mutations in the RecQ4gene, and from a healthy normal person. Northern blot analysis wasperformed on the RNAs prepared above using a probe prepared from thehelicase domain of the RecQ4 gene. The mRNAs were also probed with GAPDHas an internal control. Each lane shows the corresponding results: lane1, healthy normal person; lane 2, II.3; lane 3, AG05013; lane 4,AG05139; lane 5, AG03587; lane 6, TC4398.

FIG. 5 shows a purified partial RecQ4 protein that was synthesized in E.coli. 302 amino acids from the C terminal region of RecQ4 weresynthesized in E. Coli. The purified and dialyzed protein waselectrophoresed by SDS-PAGE and the gel was CBB-stained. The molecularweight was about 41 kD. Each lane shows the corresponding results: laneM, low molecular weight marker (1 μg); lane 1, purified protein (1 μl);lane 2, purified protein (2 μl).

FIG. 6 shows the result of Western blot analysis using normal cells andcells from RTS patients. A 100-μg aliquot of each total cell extract waselectrophoresed in a 7.5% polyacrylamide gel and subjected to Westernblotting for RecQ4. A control experiment for total amount of protein wasperformed with 10-μg aliquots of the respective total cell extracts,which were analyzed by Western blotting for actin. Each lane shows thecorresponding results: lane 1, WI38/SV40; lane 2, RTS-B (mut-1 andmut-2); lane 3, RTS-E (mut-3 and mut-4); lane 4, RTS-C (no mutation);lane 5, RTS-F (no mutation).

FIG. 7 shows an analysis for intracellular localization of RecQ4 byfluorescent antibody staining. K562 cells were attached onto a glassslide by using a Cytospin and immunostained with anti-RecQ4 antibody of2 μg/ml (A). The morphology of cells can be recognized in (B), which wasobserved in the same visual field with transmitted light.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is further illustrated in detail below withreference to the Examples, but is not to be construed as being limitedthereto.

EXAMPLE 1 Genomic DNA Cloning of the RecQ4 Helicase Gene

The genomic DNA of the human RecQ4 helicase gene was obtained byscreening a P1/PAC library. The P1/PAC library used was obtained fromGenome Systems, and the preparation method is described in the Smolleret al. reference (Smoller, et al., Chromosoma, 1991, vol. 100, pp 487-pp494). The screening was carried out by PCR using a sense primer, Q4P(5′-CGC TTC TGG AGA AAA TAC CTG CAC-3′/SEQ ID NO: 9), and an antisenseprimer, Q4Q (5′-TTG GAG CCT CCT CGT TCC CAC ACC-3′/SEQ ID NO: 10),corresponding to the base sequence segments in exon 21 of the RecQ4gene. The screening was carried out in Genome Systems Co. The isolationand purification of DNA from P1 clone #13447 obtained in the screeningwas performed by the method as descried in the reference (Smoller, etal., Chromosoma, 1991, vol. 100, pp 487-pp 494). The genomic basesequence of the RecQ4 gene was determined by using the purified P1 DNAas the template. The determined base sequence of the genomic DNAencoding RecQ4 helicase (exon 1 to exon 21) is shown in SEQ ID NO: 2.The determination of the base sequence was performed by the PCR-basedmethod described by Hattori et al. (Electrophoresis 13, pp 560-565(1992)). That is, the reaction was conducted by using a PRISM sequencingkit containing fluorescent dideoxy-terminator from Perkin-Elmer.Subsequently, the base sequence information was obtained in an automaticsequencer from Applied Biosystems (Model ABI 373), and then the data wasanalyzed by an attached Macintosh computer. RecQ4 gene-specific primersused for the base sequence determination are listed in Table 1. TABLE 1Q4 137S (5′-GTT TCC TGA ACG AGC AGT TCG ATC-3′/ SEQ ID NO:11) Q4 714S(5′-GCT GCC TCC AGT TGC TTT TGC CTG-3′/ SEQ ID NO:12) Q4 A2 (5′-TTG GTCGCA GCC CGA TTC AGA TGG-3′/ SEQ ID NO:13) Q4 A3 (5′-TGG CCC GTG GTA CGCTTC AGA GTG-3′/ SEQ ID NO:14) Q4 A5 (5′-GAC GGC TGC GCG GGA GAT TCGCTG-3′/ SEQ ID NO:15) Q4 A9 (5′-CTC AGC CCC TCC AGT CAA GCT AGG-3′/ SEQID NO:16) Q4 C5 (5′-ACC AGT GCC TCA GGT GTC AGC-3′/ SEQ ID NO:17) Q4 C8(5′-GGA AAT GTG CTG GGA AAG GAG-3′/ SEQ ID NO:18) Q4 D5 (5′-ACC AAG AGTCCA CTG CCT ACG-3′/ SEQ ID NO:19) Q4 D7 (5′-GCT CGG TGG AGT TTG ACATGG-3′/ SEQ ID NO:20) Q4 D9 (5′-AGC GCA GCA CCA GGC TCA AGG-3′/ SEQ IDNO:21) Q4 D13 (5′-GCA CTG CTT CCT GGG CCT CAC AGC-3′/ SEQ ID NO:22) Q4 E(5′-GGG TAC AGC GAG CCT TCA TGC AGG-3′/ SEQ ID NO:23) Q4 E128 (5′-CTCGAT TCC ATT ATC ATT TAC TGC-3′/ SEQ ID NO:24) Q4 F (5′-CTG GGC AGG AGCGTG CAG TCA TGC-3′/ SEQ ID NO:25) Q4 G (5′-AGG GGA GAG ACG ACC AAC GTGAGG-3′/ SEQ ID NO:26) Q4 H1 (5′-TTA GGA TCC GGG GTG CTT GTG GAG TTC AGTG-3′/ SEQ ID NO:27) Q4 H2 (5′-TTA GGA TCC CAG CTT ACC GTA CAG GCT TTGG-3′/ SEQ ID NO:28) Q4 K (5′-TCC TGG CTG TGA AGA GGC TGG TAC-3′/ SEQ IDNO:29) Q4 L (5′-ATC CCC CAA TGC AGT GCA GTC AGC-3′/ SEQ ID NO:30) Q4 U(5′-AAT CTG GGA CCT CAC TGT GAC ATC-3′/ SEQ ID NO:31) Q4 Z (5′-AGG GTGCCT TTC AGA TTG GCC TTG-3′/ SEQ ID NO:32)

The base sequence analysis revealed that the RecQ4 gene consists of 21exons and 20 introns, and its full length is about 6.5 kb.

EXAMPLE 2 Cloning of the Promoter Region of the RecQ4 Helicase Gene

DNA from P1 clone #13447, containing the genomic DNA of the human RecQ4helicase gene, was digested with restriction enzymes BamHI and BglII(TaKaRa Shuzo), and the plasmid vector pBluescriptII KS+ was digestedwith BamHI. The resulting digested DNAs were mixed with each other andthen T4 DNA ligase (TaKaRa Shuzo) was added thereto for ligationreaction. E. coli competent cells, DH5α (Toyobo), were transformed withthe reaction product and the resulting E. coli colonies were screened byPCR to determine whether or not the DNA from each colony contained a 5′upstream region of human genomic DNA of RecQ4. The screening for clonescontaining the 5′ upstream region was carried out by using a senseprimer, Q4 S (5′-TCA CAA CTT CTG ATC CCT GGT GAG-3′/SEQ ID NO: 5), andan antisense primer, Q4 R (5′-GAG GGT CTT CCT CAA CTG CTA CAG-3′/SEQ IDNO: 6), for amplifying a 247-bp segment of genomic DNA of RecQ4 sequence(residue 1399 to residue 1645). The bacteria were transferred from thecolony into a PCR reaction solution using a toothpick. The following PCRexperiment was conducted: denaturation at 95° C. for 5 minutes; 35cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C. for30 seconds, and extension at 72° C. for 30 seconds; and final extensionreaction at 72° C. for 5 minutes. After the reaction was completed, thePCR solution was analyzed by electrophoresis on a 2% agarose gel. Thecolony, in which a 247-bp band was detected, was judged to be positive.The bacteria derived from each of the resulting positive colonies werecultured in 3-ml LB medium. The alkali-SDS method was used to prepareplasmid DNA. Then the base sequence of the upstream region of thegenomic DNA of RecQ4 was determined by using the plasmid DNA as atemplate and using the following primers: Q4 A14 (5′-CAA TGG GAG GCG TCAACG TCA TCG-3′/SEQ ID NO: 7) and Q4 A15 (5′-GAG GCG AAA GAG CGG AGG GTCCAG-3′/SEQ ID NO: 8). The transcription initiation site of the RecQ4gene was previously determined by cap-site PCR (Kitao, S. et al.,Genomics, 1998, vol. 54, pp 443-pp 452; Japanese Patent Application No.Hei 9-200387). The cap-site PCR is a method for accurately determiningthe initial base in transcription. The transcription initiation site ofthe human WRN gene has also been determined by this method (Yamabe etal., Mol. Cell. Biol., 1998, vol. 18, pp 6191-pp 6200). The determinedtranscription initiation site corresponds to the first residue(residue 1) in the base sequence of genomic DNA of RecQ4 as well as inthe base sequence of RecQ4 cDNA. The base sequence of upstream region ofthe RecQ4 gene was analyzed using the obtained genomic DNA. The analysisrevealed a 5′ upstream sequence of 679 bp (SEQ ID NO: 1) from thetranscription initiation base.

EXAMPLE 3 Detection of Mutations in the RecQ4 Helicase Gene in Patientswith Rothmund-Thomson Syndrome

The inventors had previously cloned and analyzed two novel humanhelicase genes, RecQ4 and RecQ5, belonging to the RecQ helicase genefamily (see Japanese Patent Application No. Hei 9-200387; JapanesePatent Application No. Hei 10-81492; and Kitao, S. et al., Genomics,1998, vol. 54, pp 443-pp 452). Together, with these two novel genes,there are 5 members belonging to the human RecQ helicase gene family,including RecQ1 (M. Seki et al., Nucleic Acids Res. 22:4566 (1994); K.L. Puranam et al., J. Biol. Chem. 269:29838 (1994)), BLM (N. A. Ellis etal., Cell 83:655 (1995)), WRN (C.-E. Yu et al., Science 272:258 (1996)),RecQ4 and RecQ5.

Northern blot analysis for these five RecQ helicase genes revealed that,RecQ5, like RecQ1, was observed to be ubiquitously expressed through allthe tissues and organs, while markedly high level expression wasobserved in the thymus and testis and high levels in the pancreas, smallintestine and large intestine in a tissue-specific manner, like BLM andWRN for RecQ4. The fact that BLM and WRN are causative genes of Bloomsyndrome and Werner syndrome, respectively, gave the thought that theRecQ4 gene was also involved in some diseases. The present inventorsfocused on Rothmund-Thomson syndrome, which exhibits similar symptoms tothose of Bloom syndrome and Werner syndrome but for which the causativegene has not yet been identified. The inventors analyzed mutations inthe RecQ4 gene using cells and DNA derived from two patients, (brothers)II.3 and II.6, who have previously been identified and reported aspatients with Rothmund-Thomson syndrome by Lindor et al. (N. M. Lindoret al., Clin. Genet. 49:124 (1996)), cells and DNA from their parentsand cells and DNA from patients with Rothmund-Thomson syndrome unrelatedto the above-mentioned patients.

Specifically, full-length open reading frame of RecQ4 cDNA, as well asall exons of the RecQ4 gene, were first amplified by PCR from the twoRTS patients, II.3 and II.6, and their parents reported by Lindor et al.to determine and compare the base sequences.

In order to amplify the full-length open reading frame of RecQ4 cDNA,total RNA was extracted from fibroblast cell lines derived from the twoRTS patients by AGPC method (Chomczynski et al., AnalyticalBiochemistry, 1987, vol. 162, pp. 156-159), the mRNA was prepared fromthe total RNA by using Oligo(dT) 30 cellulose beads, and subsequently,cDNA was synthesized through the reverse transcription (RT) reaction.PCR for amplifying the full-length open reading frame of RecQ4 cDNA wasconducted as follows (Table 2): TABLE 2 Composition of primary reactionsolution: template DNA 1 μl 20 μM each primer (A5/A7) 0.5 μl × 2 10 ×buffer (Clontech) 2.5 μl 2.5 mM dNTPs 2 μl DMSO 1.25 μl Klen Taq.polymerase (Clontech) 0.5 μl dH₂O 16.75 μl (total volume 25 μl)Composition of secondary reaction solution: template DNA 1 μl 20 μM eachprimer (A6/A8) 0.5 μl × 2 10 × buffer (Clontech) 2.5 μl 2.5 mM dNTPs 2μl DMSO 1.25 μl Klen Tag. polymerase (Clontech) 0.5 μl dH₂O 16.75 μl(total volume 25 μl) Reaction condition:  1 × (94° C. 1 min)  5 × (94°C. 30 sec, 72° C. 4 min)  5 × (94° C. 30 sec, 72° C. 4 min) 25 × (94° C.30 sec, 68° C. 4 min)  1 × (4° C. ∞) Primer sequence A5 5′-GAC GGC TGCGCG GGA GAT TCG CTG-3′/SEQ ID No. 15 A6 5′-AGA TTC GCT GGA CGA TCG CAAGCG-3′/SEQ ID No. 33 A7 5′-CAG GTT TTG CCC AGG TCC TCA GTC-3′/SEQ ID No.34 A5 5′-GTC ACT GGC CTA GCC TCT GAC AAC-3′/SEQ ID No. 35

The resulting PCR product was excised from an agarose gel and purified.Then, the product was subcloned into a pCR2.1 vector (Invitrogen). Thedetermination of the base sequence was performed by the PCR-based methoddescribed by Hattori et al. (Electrophoresis 13, pp 560-565 (1992)).That is, the sequencing reaction was carried out using PRISM sequencingkit containing fluorescent dideoxy-terminator from Perkin-Elmer. Primersused for the determination of base sequence were as follows (Table 3):TABLE 3 Q4 A2 (5′-TTG GTC GCA GCC CGA TTC AGA TGG-3′/ SEQ ID NO:13) Q4 U(5′-AAT CTG GGA CCT CAC TGT GAC ATC-3′/ SEQ ID NO:31) Q4 T (5′-TCA TCTAAG GCA TCC ACC CCA AAG-3′/ SEQ ID NO:36) Q4 S (5′-TCA CAA CTT CTG ATCCCT GGT GAG-3′/ SEQ ID NO:5) Q4 A9 (5′-CTC AGC CCC TCC AGT CAA GCTAGG-3′/ SEQ ID NO:16) Q4 137S (5′-GTT TCC TGA ACG AGC AGT TCG ATC-3′/SEQ ID NO:11) Q4 F (5′-CTG GGC AGG AGC GTG CAG TCA TGC-3′/ SEQ ID NO:25)Q4 714S (5′-GCT GCC TCC AGT TGC TTT TGC CTG-3′/ SEQ ID NO:12) Q4 975S(5′-GGA CAC AGA CCA GGC ACT GTT GAC-3′/ SEQ ID NO:38) Q4 E (5′-GGG TACAGC GAG CCT TCA TGC AGG-3′/ SEQ ID NO:23) Q4 K (5′-TCC TGG CTG TGA AGAGGC TGG TAC-3′/ SEQ ID NO:29) Q4 H2 (5′-TTA GGA TCC CAG CTT ACC GTA CAGGCT TTG G-3′/SEQ ID NO:28) Q4 H1 (5′-TTA GGA TCC GGG GTG CTT GTG GAG TTCAGT G-3′/SEQ ID NO:27) Q4 2314S (5′-CAG GCC AGA CTC CAG GAT TGG GAG-3′/SEQ ID NO:39)

Subsequently, the base sequence information was obtained in an automaticsequencer from Applied Biosystems (Model ABI 373), and the data wasanalyzed by an attached Macintosh computer. The obtained base sequencesof the full-length open reading frames from two RTS patients werecompared with previously reported base sequence of RecQ4 cDNA (JapanesePatent Application No. Hei 9-200387) using base sequence editingsoftware, DNASIS.

Subsequently, in order to amplify exons of the RecQ4 gene from genomicDNAs, cultured fibroblast cells, which were obtained from the two RTSpatients, II.3 and II.6, and their parents, were washed with PBS, andthen suspended in TNE buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mMEDTA). Then, an equal volume of TNE buffer, containing 2% SDS and 200μg/ml Proteinase K, was added to each suspension and the resulting cellsuspension was mixed by frequently turning it upside down at roomtemperature for 1 hour. The mixture was incubated at 42° C. overnightand then DNA was extracted from the mixture. The extracted DNA wastreated 3 times with an equal volume of phenol to remove proteins.Subsequently, the sample was ethanol-precipitated to give purifiedgenomic DNA. By PCR using each genomic DNA as a template, the regioncontaining exons 9, 10 and 11 of the RecQ4 gene was amplified using asense primer, Q4 C8 (5′-GGA AAT GTG CTG GGA AAG GAG-3′/SEQ ID NO: 18),and an antisense primer, Q4 C5 (5′-ACC AGT GCC TCA GGT GTC AGC-3′/SEQ IDNO: 17); likewise, the region containing exons 13, 14 and 15 of theRecQ4 gene was amplified using a sense primer, Q4 E128 (5′-CTC GAT TCCATT ATC ATT TAC TGC-3′/SEQ ID NO: 24), and an antisense primer, Q4 D1(5′-CTC TTC ACA GCC AGG AAG TCC-3′/SEQ ID NO: 40). The following PCRreaction was conducted: denaturation at 95° C. for 5 minutes; 35 cyclesof denaturation at 94° C. for 30 seconds, annealing at 60° C. for 30seconds and extension at 72° C. for 60 seconds; and the final reactionat 72° C. for 5 minutes. The amplified DNA fragments were purified, and,using these DNAs as templates, the base sequence of the regioncontaining exons 9, 10 and 11 in the RecQ4 gene was determined with Q4C8 primer and the base sequence of the region containing exons 13, 14and 15 in the RecQ4 gene was determined with Q4 D3 primer (5′-AGA GCTGGT GTC CCC GTG GAC-3′/SEQ ID NO: 41). The determination of basesequences was performed using a PCR-based method described by Hattori etal. (Electrophoresis 13, pp 560-565 (1992)). That is, the sequencingreaction was conducted by using a PRISM sequencing kit containingfluorescent dideoxy-terminator from Perkin-Elmer. Subsequently, the basesequence information was obtained in an automatic sequencer from AppliedBiosystems (Model ABI 373), and then the data was analyzed by anattached Macintosh computer. The obtained base sequences from patientsand their parents were compared to each other by using the base sequenceediting software, DNASIS.

Based on the above-described base sequence analysis of the RecQ4helicase gene, as described below, it has been clarified that both ofthe patients with Rothmund-Thomson syndrome in this family haveheterozygous mutations. The family tree of the patients withRothmund-Thomson syndrome is shown in FIG. 1(a), and the result ofmutation analysis of in this family is shown in FIG. 1(b) and FIG. 2.

One mutation (referred to as mut-1) is present in exon 10, and it is a7-base deletion (namely, GGCCTGC of position 1650-1656 in the basesequence of protein coding region (which corresponds to nucleotide1734-1740in SEQ ID NO: 3) (See FIG. 2(a)). This deletion causes frameshit, and as a result a termination codon TGA is generated 14-basesdownstream of the deletion. Primers Q4 Cl (5′-TCT GGC CTG CCA CCG TGTCTC-3′/SEQ ID NO: 42) and Q4 C3 (5′-TGG TCA TGC CCG AGT GTA TGC-3′/SEQID NO: 43) were designed so that the mutation site of mut-1 was locatedbetween the two primers. The residue 1624-1675 in the protein codingregion of the RecQ4 gene (position 1708-1759 in SEQ ID NO: 3) (52 bp) ineach of DNAs from the parents (I.1 and I.2) and from the patients (II.3and II.6) was amplified by PCR using these primers. The resulting DNAfragment was fractionated by electrophoretic separation in a 15%polyacrylamide gel to analyze the mutations. Thus, the presence of themut-1 mutation was detected based on the difference in electrophoreticmobility (FIG. 1(b)). The analytical result showed that mut-1 wasderived from the mother.

The other mutation (referred to as mut-2) is a point mutation from C toT at residue 2269 in the protein-coding region (position 2353 in SEQ IDNO: 3). The original codon CAG (Gln) has been converted to be atermination codon TAG (FIG. 2(b)). Both of mut-1 and mut-2 havemutations in the helicase domain of RecQ4 helicase, and it is presumedthat the translation of transcripts for these defective genes isprematurely terminated, and these produce markedly smaller proteins (60kDa and 82 kDa, respectively), as compared with the molecular weight of133 kDa expected from the full length of the coding region for RecQ4helicase. The results obtained by the mutation analysis are summarizedin Table 4. The deduced truncated protein products are shown in FIG. 3.The same sequencing analysis was carried out by using DNAs prepared fromother subjects belonging to this family. In this analysis, mut-1 wasdetected in patients II.3 and II.6 with Rothmund-Thomson syndrome aswell as in I.2 cells derived from their mother; mut-2 was detected inpatients II.3 and II.6 with Rothmund-Thomson syndrome as well as in I.1cells derived from their father. That is, it was verified that mut-1 andmut-2 were derived from the mother and father, respectively, and therespective mutations had been inherited from the phenotypically healthyparents having single mutations.

In addition to these mutations specifically related to this family,another heterozygotic mutation has been found in the cell line derivedfrom a patient with Rothmund-Thomson syndrome unrelated to theabove-mentioned family. The cell line (No. AG05013) has been depositedin “Aging Cell Repository” of “National Institute of Aging (NIA)” in theUSA. The mutation in the cell line were detected by amplifying thefull-length open reading frame of RecQ4 cDNA and all exon regions of theRecQ4 gene by PCR, determining the base sequences thereof and comparingthem with the normal sequence. Procedures for amplification of thefull-length open reading frame of RecQ4 cDNA, subcloning and basesequence determination are as described above. In order to amplify exonsof the RecQ4 gene from this patient, genomic DNA was prepared fromfibroblast cells of the patient by the same method described above. Byusing the genomic DNA as a template, the region containing exons 14 and15 of the RecQ4 gene was amplified by PCR using sense primer, Q4 D3(5′-AGA GCT GGT GTC CCC GTG GAC-3′/SEQ ID NO: 41), and antisense primer,Q4 D2 (5′-TGG GAA CAC GCG CTG TAC CAG-3′/SEQ ID NO: 44). The regioncontaining exons 12 and 13 of the RecQ4 gene was also amplified by PCRwith sense primer, Q4 D11 (5′-GCC TCA CAC CAC TGC CGC CTC TGG-3′/SEQ IDNO: 45), and antisense primer, Q4 D12 (5′-GAC AGG CAG ATG GTC AGT GGGATG-3′/SEQ ID NO: 46). The condition for PCR was as described above. Theamplified DNA fragments were purified, and using the DNAs as templates,the base sequence of the region containing exons 14 and 15 in the RecQ4gene was determined with Q4 D2 primer as well as the base sequence ofthe region containing exons 12 and 13 of the RecQ4 gene was determinedwith Q4 D11 primer. The results show that one of these mutations was a2-base deletion (mut-3) and the other was a point mutation from G to Tat the boundary between intron 12 and exon 13, which destroys thesplicing donor consensus sequence (mut-4). It has been revealed thatboth mutations might cause frame shift for the translation downstream ofthe helicase domain, which respectively generate truncated proteinproducts of 881 amino acids and 794 amino acids (Table 4 and FIG. 3).TABLE 4 Rec Q4 gene mutations shown in RTS patients cell variantmutation exon situation deriviation conjugated 1650 7 bases 10frameshift Mexican- heterozygote deletion (mut-1) American C2269 (mut-2)14 nonsense mutation conjugated 2492 2 bases 15 frameshift whiteheterozygote deletion (mut-1) C2269 (mut-2) 13 frameshift

In order to clarify whether or not the patients with Rothmund-Thomsonsyndrome carrying the mutations, mut-1 and mut-2, also have mutations inthe WRN helicase gene or BLM helicase gene, poly(A)+RNAs from II.3 cellsand AG05013 cells were reverse transcribed into cDNAs and base sequenceswere analyzed by amplifying the full-length open reading frames of thecDNAs by PCR, using the cDNAs as templates. The amplified region of WRNcDNA corresponded to the residues 188-4555 of GenBank accession No.L76937 and the region of BLM cDNA corresponded to the residues 57-4370of GenBank accession No. U39817. However, no mutations were found in theWRN gene and BLM gene, which suggested that the WRN gene and BLM genewere not involved in Rothmund-Thomson syndrome. Based on the resultsdescribed above, it can be concluded that mutations in the RecQ4 geneare associated with Rothmund-Thomson syndrome. Furthermore, the resultssuggest that neither normal WRN helicase nor normal BLM helicase canrescue the deficiency caused by the mutations in the RecQ4 gene inpatients with Rothmund-Thomson syndrome.

As described above, the inventors performed mutational analysis for DNAsfrom 7 patients who had been clinically diagnosed as affected withRothmund-Thomson syndrome, and found mutations in the RecQ4 gene in 3patients including II.3 and II.6 belonging to the same family.

EXAMPLE 4 Northern Blot Analysis of the Cells from Patients withRothmund-Thomson Syndrome

To evaluate the relationship between mutations in the RecQ4 gene andpathogenesis of Rothmund-Thomson syndrome from a different viewpoint,RecQ4 mRNA from cells derived from 5 patients with Rothmund-Thomsonsyndrome were compared with that from a healthy normal person byNorthern blot analysis (FIG. 4). Total RNA was first extracted fromfibroblast cells from patients by AGPC method (Chomczynski et al.,Analytical Biochemistry, 1987, vol. 162, pp 156-pp 159), and poly(A)⁺RNA was purified from the resulting total RNA by using oligo(dT)latexbeads. The poly(A)⁺ RNA (5 μg) was electrophoresed on a 1% agarose geland then denatured with an alkaline solution. Then, the RNA wastransferred onto a nylon filter. The 321-bp fragment consisting ofresidue 2013-2333 in the RecQ4 cDNA (GenBank accession No. AB006532) wasamplified by PCR and then purified. The resulting fragment wasradiolabeled with [αa-³²P] dCTP by using a Random Primer DNA LabelingKit Ver.2 (TaKaRa Shuzo, code no. 6045) and used as a probe. The filterwas incubated in a solution containing 5×SSPE buffer, 50% formamide, 2%sodium dodecyl sulfate (SDS), 10× Denhardt's solution, 100 μg/ml salmonsperm DNA, and 1×10⁷ cpm/ml [α-³²P]dCTP-labeled probe DNA at 42° C.overnight. Subsequently, the filter was washed 3 times with 2×SSC-0.1%SDS at room temperature and then washed with 0.2×SSC-0.1% SDS at 65° C.for 30 minutes. The radioactivity was detected by autography with aBAS1500 system (Fuji film).

The results show that the level of RecQ4 mRNA of about 4 kb wassignificantly reduced in fibroblast cells derived from II.3 (lane 2), ascompared with that in fibroblast cells from healthy normal person (lane1). Such specific reduction in the level of defective mRNA has also beenobserved in the expression of WRN gene in fibroblast cells derived fromWerner patients and B lymphoblast-like cells transformed withEpstein-Barr virus (Y. Yamabe et al., Biochem. Biophys. Res. Commun.236:151 (1997)). There are a number of reports indicating that nonsensecodons influence RNA metabolism in vertebrate cells, that specificturnover of defective mRNA is stimulated and, as a result, similardownregulation of the expression can be found in other genetic diseases(L. E. Maquat, RNA 1:456 (1995); L. E. Maquat, Am. J. Hum. Genet. 59:279(1996)). On the other hand, two types of mRNAs with normal and shortersizes were detected in Northern blot analysis of mRNA prepared from theother patient (AG05013), carrying the heterozygotic mutations of the2-base deletion and the point mutation at the 3′-splice site (FIG. 4,lane 3). The short mRNA is presumed to be the product of aberrantselective splicing, due to the mutation at the splice donor site, and ispresumed to be the major molecular species for RecQ4 mRNA in thissample. On the other hand, transcripts of the RecQ4 gene, which werederived from three (lanes 4-6) of the remaining four patients withRothmund-Thomson syndrome in whom no mutations had been found in theRecQ4 gene, were essentially the same as that from normal person (lane1). These result, with respect to the transcript of the RecQ4 gene, isconsistent with results obtained in the mutation analysis of DNAsequence. Thus, it was verified that mutations in the RecQ4 generesulted in the disease in the patients with Rothmund-Thomson syndrome,II.3, II.6, and AG05013.

Diagnosis for Rothmund-Thomson syndrome on patients who are suspected tocarry this disease have been previously based on relatively broadclinical findings and, thus, has been less accurate and less reliable.It is suggested that there may exist mutations in other genes (or othergene families) in the patients, in whom no mutations had been found inthe RecQ4 gene, of the 7 patients with Rothmund-Thomson syndrome, or,alternatively, the diagnosis of Rothmund-Thomson syndrome may be wrongand in actuality the patient may be afflicted with another disease, onethat exhibits similar clinical manifestations. In addition, there is apossibility that the clinical symptoms utilized as an index for thediagnosis of Rothmund-Thomson syndrome are too broad. The disease name“Rothmund-Thomson syndrome” is often used widely for patients exhibitingsimilar but ambiguous symptoms (E. M. Vennos et al., J. Am. Acad.Dermatol. 27:750 (1992); E. M. Vennos and W. D. James, Dermatol.Clinics. 13:143 (1995)). The diagnosis of Rothmund-Thomson syndrome canbe made more accurately by utilizing gene diagnosis with the RecQ4 genesequence.

EXAMPLE 5 Preparation of Anti-RecQ4 Helicase Monoclonal Antibody

A DNA fragment containing the nucleotides 2803 to 3711 of SEQ ID NO: 3,encoding the C terminal region of RecQ4, was inserted downstream to lacpromoter/operator in an E. coli expression vector, pQE30 plasmid(QIAGEN). The plasmid DNA was transformed into an E. coli M15 straincontaining a plasmid encoding lac repressor. The resulting transformantwas cultured in LB medium (1% Bacto tryptone, 0.5% yeast extract, 0.5%NaCl, pH 7.0) containing 100 μg/ml ampicillin and 25 μg/ml kanamycin.Once the bacterial turbidity reached O.D.600=0.6-0.7, then 1 mMIPTG(isopropyl-β-D-thiogalactopyranoside) was added to the culture toinduce expression.

The E. coli was harvested by centrifugation, and then lysed bysonication in. Buffer A (50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 0.1 mM DTT,5% glycerol, 1 mM PMSF) containing 2% NP-40. Then, centrifugalseparation was repeated twice to obtain an insoluble precipitate. Theresulting precipitated fraction was suspended in Buffer A, and mixedwell with an equal volume of 1 M sucrose and 2 volumes of Percoll(SIGMA; colloidal PVP coated silica for cell separation). The mixturewas treated by ultracentrifugation (Beckman ultracentrifuge L7-65, SW28rotor, 20000 rpm, 15° C., 30 min) to yield protein inclusion body in thelowest layer. The resulting sample was washed 4 times with 50 mMTris-HCl (pH 8.0) and then dissolved in Buffer G (6 M guanidine-HCl, 0.1M NaH₂PO₄, 0.01 M Tris, pH 8.0). The Buffer G was replaced with Buffer B(8 M-1 M urea, 0.1 M NaH₂PO₄, 0.01 M Tris, pH 8.0) by dialysis, and thenfurther replaced with PBS. After the dialysis, the sample wasconcentrated by centrifugation in a conventional centrifugeconcentrator/desalting device CENTRIPLUS 10 (Amicon). The aboveprocedures provided a C terminal region (residue 2803-3711 in SEQ ID NO:3; residue 907-120 in SEQ ID NO: 4 (amino acid sequence)) recombinantprotein of RecQ4 helicase (FIG. 5).

The purified recombinant protein (50 μg), mixed with Freund's completeadjuvant, was intraperitoneally given to BALB/c mice (7-week old,female) as primary immunization. 23 days after the primary immunization,the secondary immunization was carried out by intraperitonealadministration of the purified recombinant protein (50 μg) mixed withFreund's incomplete adjuvant. 30 days after the secondary immunization,the final immunization was performed by intravenous administration ofthe purified recombinant protein (25 μg). 3 days after, spleens wasexcised from the mice. The separated spleen cells and cells of NS-1 linewere fused with each other in the presence of polyethylene glycol andsuspended in HAT selection medium. 100-μl aliquots of the cellsuspension were placed in wells of 96-well plates (560 wells in total)to cultivate the cells. In order to evaluate the antibody production inhybridomas, primary screening was performed by testing each culturesupernatant in the 560 wells according to the antigen-solid-phase ELISAmethod using the purified recombinant RecQ4 helicase protein as theantigen. The result showed that 450 wells were positive. Among thewells, 55 wells that exhibited high values measured by ELISA wereselected, and the corresponding cells were further cultured. Thesecondary screening was carried out in the same manner as in the primaryscreening according to the antigen-solid-phase ELISA method. All the 55wells selected were evaluated as positive. The top fourteen wells in themeasured values by ELISA were selected and the corresponding cells weretreated by limiting dilution method to clone the hybridomas of interest.Hybridoma clones that were evaluated positive in ELISA were establishedas monoclonal antibody-producing clones. The established hybridomas wereinoculated into BALB/c mice to prepare ascites, and purification of theantibody from the ascites was performed by the ammonium sulfate saltingmethod. A clone K6314 was selected from the resulting 14 clones and themonoclonal antibody produced by this clone was further used asanti-RecQ4 helicase antibody in the experiments described below.

EXAMPLE 6 Western Blot Analysis of Cells from Patients withRothmund-Thomson Syndrome

Western blot analysis for RecQ4 protein was carried out using humannormal cells and cells from patients with Rothmund-Thomson syndrome.Primary cultured fibroblast cells, which had been isolated from ahealthy normal person as well as from patients, were transformed withSV40 large T antigen to prepare strains of culture cells. These culturedcells were washed with PBS and then suspended in TNE (40 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 mM EDTA). The cells were harvested bycentrifugation and then suspended in Lysis buffer (50 mM Tris-HCl (pH8.0), 150 mM NaCl, 0.5% NP-40, 1 mM PMSF). The suspension was mixed byfrequently turning it upside down at 4° C. for 30 minutes. Aftercentrifugal separation, the resulting supernatant was obtained as thetotal cell extract. The concentration of protein was measured by using aProtein Assay DyeReagent Concentrate (BIO-RAD).

The prepared total cell extract was subjected to SDS-PAGE(SDS-polyacrylamide gel electrophoresis) according to the method ofLaemmli (Laemmli (1970) Nature, vol. 227, p680-685)). Proteins werefractionated on a gel by electrophoresis and then wereelectrophoretically transferred from the gel onto a nitrocellulosefilter (Imobilon transfer membrane; MILLIPORE) in a transfer buffer (20%methanol, 4.8 mM Tris, 3.9 mM glycine, 3.75% SDS) by using a TRANS-BLOTSD (BIO-RAD) at 20 V at room temperature for 1 hour. Blocking of thisfilter was carried out in PBS containing 5% skimmed milk. The filter wasincubated with the primary antibody at room temperature for 2 hours andthen washed with 0.05% Tween 20/PBS solution (PBS-T). Subsequently, thefilter was incubated with the secondary antibody at room temperature for1 hour and then washed with PBS-T. Then signal detection was carried outby using ECL Western blotting detection reagents (Amersham).

The primary antibodies used were 2 μg/ml anti-RecQ4 helicase mousemonoclonal antibody K6314 and 0.2 μg/ml anti-actin goat polyclonalantibody sc-1616 (Santa Cruz Biotechnology) in PBS solution; thesecondary antibodies were Horseradish peroxidase-conjugated anti-mouseimmunoglobulin rabbit polyclonal antibody (0.65 μg/ml; DAKO) and 0.25μg/ml anti-goat immunoglobulin rabbit polyclonal antibody in 5% skimmedmilk/PBS solution. Two bands, the molecular weight of which are about160 kD and about 140 kD, were detected in Western blot analysis of totalcell extract from normal cells (WI38/SV40) (FIG. 6, lane 1). The size,160 kD, is larger than 133 kD predicted from the number of amino acids(1208 amino acids) encoded by the RecQ4 gene, suggesting the possibilitythat the helicase is modified, e.g. phosphorylation, at the proteinlevel.

RecQ4 helicase protein was undetectable in RTS-B and E cells from thepatients with the antibody against the C terminus, as expected from theresult of mutation analysis (FIG. 6, lanes 2 and 3). Based on theabove-described results, it was confirmed that the monoclonal antibodyK6314 specifically recognizes the RecQ4 helicase protein. Further, inRTS-C and F, which are derived from patients with Rothmund-Thomsonsyndrome in whom no mutations were detected in the RecQ4 gene, RecQ4helicase protein was detected as in normal cells (FIG. 6, lanes 4 and5). These results indicate that Western blot analysis using anti-RecQ4helicase monoclonal antibody K6314 can be utilized to immunologicallydiagnose the presence of mutations in the RecQ4 gene in patients whohave been diagnosed as affected with Rothmund-Thomson syndrome.

EXAMPLE 7 Immunostaining of Cultured Cells by a Method Using FluorescentAntibody

Intracellular localization of RecQ4 protein was analyzed by fluorescentantibody staining using the above-mentioned K6314 antibody. 0.5×10⁵cells (logarithmic growth phase) of cell line K562 derived from humanchronic myelogenous leukemia were attached on a glass slide (MATSUNAMIGLASS; APS-Coated Micro Slide Glass) by using a Cytospin (TOMY SEIKO;centrifugal floating cell collector, MODEL SC-2). The cells were fixedin a solution of 3.7% formaldehyde/PBS at room temperature for 10minutes, and then washed with PBS-T (0.05% Tween 20/PBS solution). Thecell membrane permeability was enhanced in a solution of 0.1% TritonX-100/PBS at room temperature for 5 minutes. The glass slide was blockedin PBS containing 3% skimmed milk at room temperature for 1 hour andthen incubation with the primary antibody was carried out in a solutioncontaining 5 μg/ml anti-RecQ4 antibody K6314/PBS, 0.1% BSA and 0.05%NaN₃ at 4° C. overnight. The glass slide was washed with PBS-T, and thenincubaiion with the secondary antibody was performed in a solutioncontaining 7.5 μg/ml biotin-labeled anti-mouse immunoglogulin antibody(Chemicon) at room temperature for 1 hour. After washing with PBS-T, theglass slide was incubated in a solution of 5 μg/ml FITC-labeledstreptavidin (Pharmingen) at room temperature for 1 hour and then washedwith PBS-T. A solution of 2 μg/ml DAPI/50% glycerol was used to mountthe sample and was counterstained for chromosomes by DAPI. Microscopicexamination was carried out with an Olympas laser scanning biologicalmicroscope, FLUOVIEW system BX50.

In this observation, RecQ4 protein was detected as a very fine grainover the entire nucleoplasm (FIG. 7). This result suggests that theRecQ4 protein functions in the nucleus and also that the K6314 antibodyis useful in the analysis of the functions of RecQ4 helicase.

INDUSTRIAL APPLICABILITY

The present invention reveals that Rothmund-Thomson syndrome is agenetic disease caused by mutations in the RecQ4 helicase gene. Thisfinding makes it possible to conduct diagnostic tests forRothmund-Thomson syndrome, including diagnose of a disease asRothmund-Thomson syndrome and prenatal diagnosis for Rothmund-Thomsonsyndrome, and to perform treatments for Rothmund-Thomson syndrome,including gene therapy, by utilizing the RecQ4 helicase gene, primers orprobes designed based on the sequence thereof, RecQ4 helicase, andantibodies thereto.

1. A method for the diagnosis of Rothmund-Thomson syndrome,characterized by detecting mutations in the DNA encoding RecQ4 helicaseor the expression regulatory region thereof.
 2. The method for thediagnosis of Rothmund-Thomson syndrome in claim 1, comprising the stepsof: (a) preparing DNA samples from patients; (b) amplifying the preparedDNA samples primers for a DNA encoding the RecQ4 helicase or theexpression regulatory region thereof, and determining the base sequence;and (c) comparing the determined base sequence with that of a healthynormal person.
 3. The method for the diagnosis of Rothmund-Thomsonsyndrome in claim 1, comprising the steps of: (a) preparing RNA samplesfrom patients; (b) separating the prepared RNA samples according totheir size; (c) hybridizing a probe for an RNA encoding the RecQ4helicase to separated RNAs; and (d) detecting the hybridized RNA andcomparing the results with that of a normal, healthy person.
 4. Themethod for the diagnosis of Rothmund-Thomson syndrome in claim 1,comprising the steps of: (a) preparing DNA samples from patients; (b)amplifying the prepared DNA samples using primers for a DNA encoding theRecQ4 helicase or the expression regulatory region thereof; (c)dissociating the amplified DNA into single-stranded DNA; (d)fractionating the dissociated single-stranded DNAs on a non-denaturinggel; and (e) comparing the mobility of the fractionated single-strandedDNA on the gel with that of a healthy normal person.
 5. The method forthe diagnosis of Rothmund-Thomson syndrome in claim 1, comprising thesteps of: (a) preparing DNA samples from the patient; (b) amplifying theprepared DNA samples using oligonucleotides comprising a base that formsa base pair with the mutated base specific to Rothmund-Thomson syndromein the DNA encoding RecQ4 helicase, or the expression regulatory regionthereof, as at least one of the primers; and (c) detecting the amplifiedDNA fragment.
 6. The method for the diagnosis of Rothmund-Thomsonsyndrome in claim 1, comprising the steps of: (a) preparing DNA samplesfrom patients; (b) amplifying the prepared DNA samples using a pair ofprimers for a DNA encoding the RecQ4 helicase or the expressionregulatory region thereof which flank the mutated base specific toRothmund-Thomson syndrome; (c) hybridizing to the amplified product apair of oligonucleotides selected from the group of: (i) anoligonucleotide synthesized such that the base forming a base pair withthe mutated base in the amplified product corresponds to the3′-terminus, and an oligonucleotide synthesized such that theneighboring (on the 3′side) base to said 3′-terminus corresponds to the5′-terminus; (ii) an oligonucleotide synthesized such that the baseforming a base pair with the base of a normal healthy person whichcorresponds to the mutated base in the amplified product corresponds tothe 3′-terminus, and an oligonucleotide synthesized such that theneighboring (on the 3′side) base to said 3′-terminus corresponds to the5′-terminus; (iii) an oligonucleotide synthesized such that the baseforming a base pair with the mutated base in the amplification productcorresponds to the 5′-terminus, and an oligonucleotide synthesized suchthat the neighboring (on the 5′ site) base to said 5′-terminuscorresponds to the 3′-terminus: and (iv) an oligonucleotide synthesizedsuch that the base forming a base pair with the base of a normal healthyperson which corresponds to the mutated base in the amplified productcorresponds to the 5′-terminus, and an oligonucleotide synthesized suchthat the neighboring (on the 5′ site) base to said 5′-terminuscorresponds to the 3′-terminus; (d) ligating the oligonucleotides: and(e) detecting the ligated oligonucleotide.
 7. The method for thediagnosis of Rothmund-Thomson syndrome in claim 1, comprising the stepsof: (a) preparing protein samples from patients; (b) contacting anantibody against RecQ4 helicase with the prepared protein sample; and(c) detecting proteins binding to said antibody.